University of ConnecticutDigitalCommons@UConn

Honors Scholar Theses Honors Scholar Program

July 2006

The Nature of Genetic Engineering and the Usesand Potential Abuses of BiotechnologySeth J. MattonUniversity of Connecticut, SETH.MATTON@huskymail.uconn.edu

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Recommended CitationMatton, Seth J., "The Nature of Genetic Engineering and the Uses and Potential Abuses of Biotechnology" (2006). Honors ScholarTheses. 21.http://digitalcommons.uconn.edu/srhonors_theses/21

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The Nature of Genetic Engineering and the Uses and Potential Abuses of Modern

Biotechnology

By Seth Matton

Thesis Advisor: Dr. Wolf Dieter-Reiter, Ph.D

Honors Scholar Thesis

Department of Molecular and Cellular Biology University of Connecticut

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Table of Contents

I) Introduction 1

II) Genetic Engineering: Using Nature’s Tools 2-13

III) Human Health and Genetic Engineering

A) Addressing Fears about Biotechnology and Human Health 14-25

B) Improvements to Human Health: Genetic Engineering and Medicine

25-31

IV) Genetic Engineering and the Environment

A) Environmental Benefits of Genetic Engineering 32-40

B) Addressing Fears about Biotechnology and the Environment 40-48

V) Bioterrorism 49-53

VI) Conclusion 54

VII) References 55-56

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Abstract: Over the past decade the topic of genetic engineering has been has been readily debated in the media, but often these debates consist of political rhetoric and fail to offer objective information on the methods and the potential benefits to human health and their environment. In truth, humans have been manipulating the genomes of organisms for thousands of years, and it has been an evolution of scientific knowledge that has led to the more precise methods of genetic engineering. This paper discusses how scientists utilize natural processes to alter the genetic constituents of both prokaryotic and eukaryotic organisms, benefits to human health and the environment, as well as potential misuses of biotechnology such as bioterrorism.

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I) Introduction

Some have argued that genetic engineering is unnatural, that humans are forcing

the hand of God by tinkering with genes and making products that would have never

been produced if nature had been left alone. However, humans have been using

organisms to produce certain products and selectively steering evolution since the

beginning of civilization. In fact, one can argue that human intervention into biological

processes has allowed civilization to form in the first place. Yogurt and beer have been

made for centuries using the metabolic pathways of organisms. Yet nobody has criticized

Yoplait or Budweiser of playing God. Similarly, animals have been selectively bred to

produce desired characteristics, such as breeding wild wolves into dogs and wild oxen

into passive dairy cows, yet people still drink milk and there are no rallies against puppy

dogs. Genetic engineering is merely an extension of a process that began at the advent of

the agrarian society as a means of survival. Legitimate concerns can be voices against

widespread use of recombinant genetic technology. Unfortunately, too often the loudest

protests are merely knee-jerk diatribes of what they think (or fear) genetic engineering to

be, which is certainly not a fair or credible discussion, and completely fails to

acknowledge the amazing potential that this burgeoning technology can bring to

mankind. This paper is an honest discussion of just how natural genetic engineering is,

the technology used, the potential problems of wide scale application of the technology,

as well as the solutions that scientists have devised to answer these problems.

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II) Genetic Engineering: Using Nature’s Tools

Although a discussion of what “natural” means falls outside the realm of

molecular biology, it seems to be a necessary discussion since the supposed

unnaturalness of genetic engineering is a sticking point in most people’s minds. Bad

Hollywood movies and large public relations campaigns have blurred what exactly

modern science is trying to do. Nevertheless, a definition of “natural” is more elusive

than it may seem. If a number of people on the street were asked whether natural was

good, chances are all of them would say yes. Yet if they are pushed for a definition of

what exactly “natural” means, they would probably struggle at first, and then after several

attempts probably come up with something along the lines “as God intended,” or “as

nature created things.” Natural is universally thought of as good, no matter how difficult

its definition is to pin down. On the other hand, topics such as genetic engineering (or

perhaps any other modern technology) has always been met with unease. There has

always been a hasty reaction to resist scientific progress. It conjures up the archetypal

image of Adam eating from the tree of knowledge, that something sinister lurks behind

the thrust of progress. Perhaps the concepts of natural vs. unnatural can be more

accurately distilled as rational vs. irrational. The concept of “natural” seems to be a

refuge people take from the rational; an oasis to people who feel an impulse to interpret

the causes of events as mysterious, divine, and unknowable.

It is extremely easy to disprove the argument that natural is good, and when

humans tinker with nature, it’s bad. This is a naïve judgment. In fact, it’s a complete

misinterpretation of how life perpetuates itself through evolution. Evolution doesn’t

particularly care specifically about human life, or for that matter, any form of life over

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another. Organisms try to survive, and if by chance they are allowed to survive and

reproduce more efficiently than other organisms, then they have a decided advantage, no

matter if they are bacteria, insects, or humans.

It is one of the most cited fears of genetic engineering that an organism will

acquire a resistance to an insecticide, herbicide, or even an antibiotic marker gene used to

test for transformation, and this will lead to a "superbug" that is resistant to all known

methods of suppression. Bacteria obtaining resistance to all prescribed forms of

antibiotics and causing widespread human illness and death is a worry in many people’s

minds. This, of course, is possible, but antibiotic resistance is hardly a phenomenon

introduced by genetic engineering. There are probably thousands of these “superbugs” in

most people’s back yards. A January 2006 article in Nature [1] describes an experiment

where a team of microbiologists collected random samples of dirt from towns and forests

around Canada, and isolated 460 different strains of Steptomyces, a common soil

bacterium On average the strains were unaffected by seven of eight antibiotics (both

natural and synthetic varieties) commonly prescribed for bacterial infections, with two

strains being resistant to 15 drugs! [2]. There are superbugs all over the place!

Most of these microorganisms were found far from farms and hospitals, so there

is no possibility that they evolved antibiotic resistance due to human activity. Rather,

these microorganisms may simply lack transport mechanisms to take up the antibiotic,

which renders them impervious to these molecules. Of course, Streptomyces species are

known to secrete antibiotics naturally, perhaps to ward off any other bacterial species that

may try to invade their area and resources, so it is not surprising that there are numerous

species found that won’t be affected by antibiotics. If they were affected, they wouldn’t

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have been permitted to survive in the first place.

The central problem with the “natural is always better” viewpoint is that it’s an

egocentric viewpoint, as if nature exists to serve man. Unfortunately for us, all organisms

try to survive, and sometimes they survive at the expense of human life. Yersinia

pseudotuberculosis is a bacterium which causes mild intestinal disorders to animals and

humans who are infected by it. Centuries ago, long before the advent of genetic

engineering, the genes encoding adhesion proteins which once allowed Yersinia

pseudotuberculosis to adhere to the intestinal walls of the infected animal were altered

[3]. This alteration, although very slight, gave this organism a decided advantage over

non-mutant varieties. Of course, this minor mutation occurred merely by chance, perhaps

due to UV damage affecting its genome. Now these altered bacteria not only had the

ability to infect the intestines, but also move to other organ systems and cause more

severe complications, causing death to 1 in 4 of every human being infected. The altered

bacterium is called Yersinia pestis, which is the culprit that caused the plague in Europe

during the Middle Ages. This is an instance of nature producing something that wasn’t so

good for human beings.

It may seem like getting a plant or other organisms to produce a non-native

protein is a radical feat, as if humans are playing God with nature, but in fact scientists

are only using the tools that organisms gave them. Humans didn’t invent restriction

enzymes to cut a specific position along an organism’s genome. Bacteria naturally

evolved restriction enzymes to cut foreign DNA from invading viruses. Of course, this

ability to cut DNA would be useless without the ability to paste the genes in the desired

location. This is the job for ligase, which again is naturally produced by all cells. Each

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living cell contains this ligase enzyme to reconnect the sugar backbone of our DNA in the

event that our DNA polymerase made a mistake during replication and had to remove a

base. Once again, the tools for genetic engineering weren’t invented by scientists.

Scientists merely use the processes that the natural world provided them.

In fact, the natural abilities of the organisms perform all the work in genetic

engineering, and the scientists merely tweak the processes. A good example is

Agrobacterium tumefaciens, which is a soil bacterium that infects dicotyledonous plants

such as grapes, peaches, and roses, eventually causing crown gall tumors [6]. It does so

through the naturally occurring Ti plasmids, that allows a portion of its DNA

(T-DNA) to insert into a plant’s genome through illegitimate recombination. Among the

essential sequences on this plasmid are left and right borders that specify where the

plasmid is to be cut to yield the single stranded piece of T-DNA, as well as numerous

virulence genes which encode the complex machinery that performs the conjugation

process. There are also genes on the T-DNA segment that code for the synthesis of

opines. These genes show how brilliant evolution is in furnishing organisms unique

niches for survival. Opines are a carbon source only for A. tumefaciens, and can’t be used

by plant cells, or even other soil bacteria living in the environment. Essentially A.

tumefaciens infects a plant with its extrachromosomal plasmid, and uses the plant as a

factory to make nutrients so it can survive. This is an ingenious mechanism that is also

harnessed by scientists. If bacteria can use plants for biosynthetic factories, humans

should be able to as well. And in fact they do.

Perhaps the most commonly used product of genetic engineering, the Bt

insecticide, is introduced into plant cells using a modification of the mechanism used by

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A. tumefaciens. A variety of genes can be inserted in the T-DNA region of a Ti plasmid.

This DNA can then be inserted into the plant genome, and use the plant’s biosynthetic

machinery to make the proteins encoded by these genes. This system came about long

before genetic engineering. Slight modifications can be made by the scientist to cut out

unwanted genes (such as the auxin, cytokinin, and opine genes) as well as other

manipulations to get the Ti plasmid system to work in a wider range of plants than just

dicots (the methodology is covered in more depth later in the paper).

However, the broader message is that exchange of genetic material is a common

feature of many organisms in nature. Without it life wouldn’t have evolved to what it is

today. In fact, humans wouldn’t have evolved at all if it weren’t for some genetic cutting

and pasting. Opponents of genetic engineering frequently complain that strange and

unnatural genes are being inserted into our foods—the genetic purity of our natural

environment is being disturbed. This is a naïve belief. Genetic purity simply doesn’t

exist. The notion of genetic purity is actually antithetical to how the complexities of life

came about. Human beings are a prime example. DNA sequences that are viral in origin

make up nearly half of our genome [7]. Viruses are the best genetic engineers that nature

has, especially retroviruses, which on occasion can infect a germ cell and be passed on

from generation to generation like any other gene. Although most people associate

viruses with such nuisances as the common cold, or fatal diseases such as HIV and Ebola,

viruses have provided a major driving force in the evolution of all life, including many

beneficial traits. Gene expression, polyadenylation of mRNA, genomic recombination,

and pregnancy are all influenced by these viral fragments in our genes [8]. These

sequences continue to be a potent force for genetic change in human evolution. Since

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retrotransposons are essentially retroviruses in our own genomes, an RNA copy can be

made after the initial integration of the viral sequence, and using the reverse transcriptase

encoded in the sequence, can make a complementary DNA sequence that can integrate

into another place in the genome. It is estimated that there are at least 1 retrotransposition

event in every 50 sperm [11], which potentially can create and introduce new regulatory

regions or genes into the human gene pool.

Most of these HERV (human endogenous retrovirus) sequences are non-coding

due to accumulation of mutations or deletions. However, it has been found that 18 fully

coding envelope genes of retroviral origin are produced in healthy tissues, including two

recent finds that some of these genes assist in cell to cell fusion during the

synciotrophoblast formation during pregnancy [8]. In fact, three separate sequences that

were once retroviruses supply vital components of human pregnancy and fetal

development [9]. Virally derived genes control the immunosuppressant characteristics of

the placenta, so the mother’s immune system doesn’t attack the developing fetus, as well

as cell fusions so that the placenta can implant on the mother’s uterine walls [8].

The LTR (long terminal repeat) of one of these segments, named ERV3, also

contains several sex-hormone cis response elements such as a progesterone control

element [8]. Since progesterone controls trophoblast proliferation, the presence of viral

elements plays an essential role in early development. This is an example of a viral

remnant that doesn’t necessarily exist in a coding region, so it isn’t a gene, but

nonetheless provides a regulatory region that influences the expression of a gene. There

exists a vast potential for these sequences to control gene expression and perform

regulatory functions in the nearly 98.5% of the human genome that doesn’t code for

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proteins.

A detailed discussion of the influence of retroviruses on human evolution would

take thousands of pages. The bottom line is that nature cuts, pastes, mutates, inverts, and

makes numerous alterations to the genomes of every organism, and it is these fortuitous

alterations that provide the driving force of evolution. What humans do in genetic

engineering is mild compared to the power that microorganisms possess. Once again,

humans are only using some of the vast number of tricks that organisms already possess

to alter genetic information. In this respect, genetic engineering is a poor term to describe

the work being done on genetic material, since it suggests that humans can alter

organisms in whatever ways they desire, as if they could (or desire to) make strange and

exotic creatures at will. It would surprise some opponents of genetic engineering just how

limited scientists are in their actions. The introduction of genes into the correct position

in another organism’s genome is not a simple or precise activity, and even if the genes

are integrated, gene regulation is far more complex than once expected. A gene can’t

simply be introduced into another organism and be expected to produce the correct

product in the right quantities for a particular cellular function. Metabolic pathways are

also complex, and there are specific characteristics of the host organism, such as its

endogenous pool of tRNA’s, that may limit the amount of a gene product that is

produced.

Whatever complexities may arise in the manipulation of organisms, it is clear that

the desire to alter organisms to perform a particular function in not a new one. It is borne

out of the instinct for survival. This same instinct drove humans to domesticate wild

animals, selectively breed wild weeds to produce nutritious crops, and to continue to

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pursue scientific knowledge. We do all these things so we are less vulnerable to the

seemingly random shifts in our environment that are caused by a near infinite amount of

factors that are far beyond the control of humans.

In fact, in many instances humans are at a severe disadvantage in the fight for

survival. Bacteria can replicate at a much faster rate. “Humans would require a

millennium to produce the same number of generations that a bacterium can in a single

day” [7]. This leads to much fewer humans, as well as fewer opportunities for genetic

mutations that can lead to beneficial traits. Also, humans can’t transfer genes from

individual to individual like bacteria can. Three known processes exist in which one

bacterium can horizontally transfer its genetic information to other bacteria: transduction,

conjugation, and transformation. Transduction is the movement of genetic information

via bacteriophage, which infect one bacterial cell, replicate their genome, then somewhat

imprecisely excise themselves, sometimes taking a chunk of the host cell’s genome with

them. Then following the bursting of the host cell, the bacteriophage can infect another

bacterial cell, now carrying a portion of the previous cell’s genome with it. The second

method is conjugation, which involves cell to cell contact and the transfer of a plasmid

among two bacterial cells. The third process is transformation. This involves the uptake

and incorporation of naked DNA between bacterial cells.

Obviously, humans can’t freely swap genes from one person to another. This,

added to the fact that our generation time is much longer than the generation time of

microorganisms, makes us decidedly less fit for survival. However, perhaps the most

important quality that gives humans an advantage over all other organisms is our

intellect. In this respect, intelligence is an advantage that evolution has afforded us, so

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any activity of that intellect can be seen as an extension of evolution. And since genetic

engineering is a product of our intellect, and intellect is a means of survival for us just

like swapping DNA is a means of survival for bacteria, it doesn’t make sense to forfeit

our selective advantage and not use our intelligence to alter the biological world in our

favor to give ourselves any advantage we can get. Again, because humans are at a

disadvantage in so many aspects of life, not doing everything we can to survive is

antithetical to evolution. Pathogenic bacteria aren’t going to forfeit the ability to infect us

and our food sources, so we shouldn’t forfeit the ability to increase crop yields, protect

our food sources, make cheap and reliable vaccines, and the numerous other benefits that

genetic engineering can provide us.

Besides, the manipulation of the genetic material of organisms is not a new

activity for man. Ever since the start of primitive societies, humans began to tinker with

the DNA of different organisms, all toward the accomplishment of a particular aim. The

cows grazing in the pastures of today’s farms were once wild oxen, but through selective

breeding were “engineered” to be large, lazy and perfect creatures for the production of

milk and meat. Wild boars were engineered into pigs, wolves were engineered into dogs,

and all the animals that serve some function in modern societies were once wild animals

that were selectively bred to serve our needs. Now the definition of genetic engineering—

the tinkering with an organism’s genes to produce a desired outcome—seems to have

been practiced for some 10,000 years. The desire was the same then as it is now. The

only difference is that our tools for executing that desire have improved tremendously.

This is also a point where the argument that genetic engineering is “unnatural”

breaks down. One cannot simply say that natural is the way the world would be if man

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hadn’t tinkered with it, or look back to previous generations and claim those societies

lived more naturally, since the scaffolding of even the most ancient societies was based

on the manipulation of the genes of the organisms around them. All the major crops

around the world were once weeds, incapable of feeding more than a small group of

people at a time. “Most people who are concerned about modern biotechnology have

little or no knowledge of the processes that were used to transform crops in the past. Nor

are they likely aware that crops have been continually altered over time or that, without

human care, they would cease to exist” [4]. If plants weren’t crossed, recrossed with wild,

related species and selectively bred over a period of thousands of years, potatoes and

apples would be smaller than the size of a marble! “Today’s wheat, even before the

advent of genetic engineering, differs from its ancestors in so many ways that it is

classified as a different species” [3].

Figures from Prakash, Channapatna. (2001) “The Genetically Modified Crop Debate in the Context of Agricultural Evolution. Plant Physiology. 126, 8-15.

Figures 1 and 2 shows a native corn and tomato before and after humans began to

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selectively breed these species. These phenotypic traits are the result of humans

purposefully altering the genotypes of these plants. Although humans in ancient societies

had no idea that DNA was the source of the phenotypic changes they sought, they

deliberately guided evolution to produce crops that could sustain large groups of people

in a stable residence, thus making them less vulnerable to the myriad of factors that could

compromise their survival. It was crude genetic engineering. But without it we would still

be primitive creatures, wandering from area to area wherever naturally occurring foods

are plentiful, leaving when the source has been diminished, and perpetually vulnerable to

every frost, flood, drought, infestation—every sudden change in environment that’s an

inevitable part of a large, complex ecosystem. Without man’s manipulation of the genetic

world around him, a historical consciousness wouldn’t have even developed to a point of

questioning what is natural and what is not.

Hundreds of pages can be written to show how natural modern genetic

engineering really is. However, the undisputable difference between the processes that

occur in the laboratory and those that occur in the wild can be succinctly listed on a

single page. First of all, evolution in the wild takes a much longer time to alter the genes

of a species. This is something of an understatement. Evolution may take hundreds of

thousands to millions of years to alter a species, whereas in a laboratory this can take

only weeks. Modern genetic engineering can be described as speedy, controlled

evolution. In fact, it’s more accurate, controlled evolution compared to traditional

breeding. If a farmer tries to get a particular trait expressed in one of his plants, he must

cross two of the plants with the desired traits, wait, cross again, wait, and repeat this

procedure many times until the desired trait is expressed in all of the generations, if the

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traits can be expressed at all. This traditional breeding method is not specific, introducing

perhaps thousands of genes at a time. A farmer using the conventional methods may

achieve resistance to a disease, for instance, but also introduce a variety of unwanted

genes that may be potential allergens or toxins. On the other hand, genetic engineering

allows a specificity that can’t be matched by traditional methods. A single known gene

can be inserted into another organism’s genome and be controlled by a promoter that

allows the gene to be activated when desired. Genetic engineering is as natural as

traditional breeding methods, but more efficient, more accurate, and safer. Modern

scientists use nature in a more intelligent way than past generations.

Secondly, for the most part there is a species barrier in the movement of genetic

information in the wild, with the rare exception of gene movement through the infection

by viruses. However, in the laboratory the species barrier has been knocked down. Now

animal genes can be expressed in bacteria and plants (and vice versa), and a gene from

one species of animal can be expressed in another species of animal. Of course, this

manipulation of genes in the laboratory requires an in vitro step, followed by the target

organism carrying out the brunt of the work in vivo, whereas evolution carries out its

business completely outside of the test tube.

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II) Human Health and Genetic Engineering A) Addressing Fears about Biotechnology and Human Health

Another common protest against genetically altering crops is that it will produce

synthetic, unsafe products that will cause damage to human health in the long term.

People have even called genetically modified food crops “Frankenfood,” as if scientists

are huddled away in dank basements concocting strange substances that will have

horrible and unforeseen consequences for humankind.

Once again, those who protest against genetic engineering generally don’t

understand the principles by which transgenic foods are made. First of all, just because

something is produced through natural breeding methods doesn’t mean it is necessarily

healthy. Secondly, nature has produced a huge variety of plants that just so happen to be

hazardous to human health. “Our daily food naturally contains thousands of chemicals,

and many of them are shown to be carcinogens or hazardous in lab animal studies with

huge doses. We consume roughly 5,000 to 10,000 natural toxins daily, as plants have

evolved to produce an array of chemicals to protect themselves against pests, disease, and

herbivores” [4].

Genetic engineering has an enormous potential to enhance the nutritional contents

of crops and other organisms. By altering one or a few select genes, an organism that

once provided little or no nutrients can be stimulated to produce a valuable product that

can serve to fulfill nutritional needs for humans. Of course this is not a new activity.

Thirty years ago, traditional breeding was used to select rapeseed plants which didn’t

produce the nutritionally undesirable erucic acid, but instead produced canola oil, which

has much greater health benefits to humans [5]. Now canola oil is so commonly used that

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it supplies a large proportion of fatty acids consumed in most developed nations. Flax

seeds were similarly bred to produce edible high linolenic acids similar to those produced

in corn oil, instead of high linolenic oils used for industrial purposes [5].

As impressive as this was, genetic engineering can make even greater advances in

nutritional value than traditional breeding. It does not require a large overhaul of the

organism’s genome to get it to produce a desired product. Specific organisms are

selected that naturally synthesizes an intermediate or possess certain biosynthetic

pathways, so only slight modifications are needed to produce a new product. It should

console the opponents of genetic engineering to know that the alterations scientists make

to organisms are slight, and it is the natural ability of the organism that provides most of

the work. Transgenic plants aren’t Frankensteinish creations, but ordinary plants that

differ by as little as a single gene. And while these alterations pose virtually no risk to

human health, the potential benefits are amazing.

Take for example “Golden Rice.” Rice is a staple in the diets of billions of people

around the world, especially those in developing countries. Unfortunately the oil-rich

aleurone layer of rice is removed to allow it to be stored for longer periods of time

without going rancid, leaving behind the starchy endosperm containing few essential

vitamins [12]. By adding two genes coding for enzymes in the β-carotene biosynthetic

pathway, an essential nutrient for humans can be produced by the rice endosperm that

would have never been able to be achieved through traditional breeding.

While vitamin deficiencies have been eradicated decades ago in America and

other first-world countries, there continues to be millions of people around the world that

can’t obtain nutrients in order to achieve basic health. For instance, inadequate supply of

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vitamin A is a serious public health concern in at least 26 countries, including highly

populated countries in Asia, Africa, and Latin America [12]. More than 250,000 children

go blind every year in Southeast Asia due to retinol deficiency [3]. This is an instance

where poor countries not only lack the luxury of modern medicines and technologies, but

lack the essential factors to survive. More than 125 million people in the world

experience vitamin A deficiency [3]. Yet it only takes the addition of two genes to

potentially protect millions of people from the crippling effects of vitamin A deficiency.

Rice is a perfect target for this engineering for a couple of reasons. First of all,

rice is plentiful and relatively cheap to grow in these poor countries. Secondly, the rice

endosperm synthesizes an early intermediate in the beta-carotene biosynthetic pathway,

called geranyl diphosphate, that can be acted on by the enzyme phytoene synthase to

produce carotene phytoene, which is the next substrate in this pathway [12]. The product

is a provitamin form of vitamin A, which is cleaved in the body to produce retinal, the

usable form of vitamin A.

From Beyer, Peter et al. “Golden Rice: Introduing the β-Carotene Biosynthesis Pathway into Rice Endosperm by Genetic Engineering to Defeat Vitamin A Deficiency.” J. Nutr. 132:506S-510S (2002).

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It’s an extremely simple answer to a major problem. However, the “Golden Rice”

experiment came under harsh criticism because it yielded lower amounts of β-carotene

than expected. Some critics even went as far as to claim that the scientists performing the

experiment committed fraud. It’s unfortunate that people tend to cast off an experiment as

a failure simply because a single attempt failed to produce miraculous results. The

demand for immediate and ideal results is hardly ever satisfied in science, since any kind

of groundbreaking work is a process, one which takes the cooperative efforts of groups of

scientists and constantly evolving protocols to achieve the desired results. The “Golden

Rice” experiment was a success, although it may not have produced the amounts of β-

carotene that some people expected. However, just a few years later the yield of β-

carotene was greatly increased by a simple adjustment.

It was discovered a few years later that the step limiting the accumulation of β-

carotene in the rice endosperm was the phytoene synthase originating from Narcissus

pseudonarcissus [13]. Instead, the phytoene synthase from maize was found to be more

effective. When added to the carotene desaturase from the original golden rice

experiment, β-carotene accumulation increased up to 23 fold! A maximum yield of 1.6

mg/g of carotenoids was achieved in the original experiment, compared to 37 mg/g by

using the maize phytoene synthase [13]. Based on the current RDA, 50% of a child’s

daily requirements for vitamin A could be satisfied by the β-carotene in 72 grams of the

golden rice made in this experiment [13].

It’s clear that genetic engineering is far superior to traditional breeding in

enhancing and adding nutrient content to foods. Genetic engineering can even make

foods that were once unhealthy a little better for us. Researchers have actually found a

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way of making bacon healthy [14]. Nuclear transfer cloning was used to insert the fat-1

gene into pig primary fetal fibroblasts, allowing pigs to convert the abundant omega-6

fatty acids into omega-3 fatty acids, which are the unsaturated fatty acids that have been

shown to improve cardiovascular health. This breakthrough will allow scientists to

further study the health benefits of omega-3 fatty acids, as well as giving people an

alternative source for these healthy fatty acids besides fish (which contains mercury as

well). Once again this engineering was only a slight alteration, adding a single gene—the

fatty acid desaturase gene—which pigs naturally lack.

If genetic engineering can help prevent blindness and even make eating bacon

healthier, who in their right mind would protest against it? If people objectively examine

the facts they would come to the conclusion that the potential benefits far outweigh the

potential risks. Unfortunately, every time a new technology or a new paradigm about

doing things is introduced, there is always the initial apprehension, even sometimes

outright revolt. However, if the changes don’t lead to harm, or if the changes are so badly

needed that it becomes a necessity, then acceptance follows and the changes become the

norm. People in the United States have been eating genetically modified foods for several

years now, yet there hasn’t been a mass outbreak of allergic reactions or other health

problems. If you’ve consumed breakfast cereals, junk foods, bread, etc in America in the

past five years, then chances are that you’ve consumed genetically modified food.

Genetically modified ingredients are present in 60 to 70% of supermarket products in the

United States [15]. In 2003, 81% of the soybean crops in America were genetically

modified, and many soybean based ingredients such as oil, flour, lecithin, and protein

extracts are added to most processed foods [15].

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While only 40% of corn crops in America were genetically modified in 2003,

genetically modified and non-genetically modified corn are not separated by growers and

processors, so if someone has consumed a corn-based product, then they have

undoubtedly consumed a genetically modified product [15]. Yet there hasn’t been a mass

rush of people to emergency rooms with allergies against new protein in foods. In fact, it

can be said that a massive practical experiment has been performed on the American

people over the past several years. Millions of people have consumed the thousands of

products on supermarket shelves which contain at least some genetically modified

ingredients in them, and there have been no evident health effects. Most people didn’t

even realize they were consuming foods that were genetically modified.

Clearly these foods are safe. But this is not a coincidence. Genetically modified

foods are the most stringently tested foods on the market. The figure below is an example

of all the tests performed on a new food before it enters the marketplace.

Fig 3

From World Health Organization. “Safety Aspects of Genetically Modified Foods of Plant Origin: Report of a Joint FAO/WHO Expert Consultation on Foods Derived from Biotechnology.” (2000)

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Any transgenic crop engineered with pesticidal traits is closely examined by three

different government agencies: the Environmental Protection Agency (EPA), the US

Department of Agriculture (USDA) and the Food and Drug Administration (FDA) [17].

All three organizations closely scrutinize new products to make sure that they are safe for

the environment and human health.

The FDA requires that each company proposing a new transgenic product for

public consumption do their own stringent independent testing to determine any potential

allergic reactions before these products are even considered for sale. Normally in vitro

digestion assays are performed (to see if the product will remain in the digestive system),

amino acid sequence comparisons to known allergens are made, and acute oral toxicity

tests are done with large doses of the purified transgenic protein in question [17]. The last

test involves the ingestion of the purified protein in mice at 3,280 to 5,000 mg per kg of

body weight [17]. It should be noted that only the protein that is added by the new gene is

administered in these trials, and not the complete product, in which only a small fraction

of the total protein is transgenic. Of course, these tests administer far more of the added

protein than could even be consumed in the normal diet. Government agencies like the

FDA err on the side of caution, so that anything short of falling into a vat of purified

protein and having to eat your way out will be safe.

There have been examples where the FDA stopped a product from being

produced because it was suspected of containing a known allergen. The structure of any

novel protein that may appear in food is compared to the structure of common allergens,

and anything showing amino acid sequence similarities to known allergens is extensively

24

scrutinized. When a genetically modified soybean was suspected of containing an

allergen, it was never allowed to see the grocery store shelf. Soybeans offer many

nutritional benefits, but their nutritional value is somewhat compromised by the fact that

they lack substantial levels of methionine.

Traditional breeding methods can be used to increase the total amount of protein

in the soybean, and site directed mutagenesis in conjunction with traditional breeding

may be able to increase the production of sulfur-containing amino acids, but the most

realistic and efficient way to get a soybean to produce methionine is to genetically

modify it [18]. This is exactly what was done. The methionine-rich 2S albumin gene from

the Brazil-nut was introduced into the genome of soybean [19]. When subjects with

Brazil-nut allergies became allergic to the transgenic soybean but not the regular

soybean, it was a clear indication that the major allergen in the Brazil-nut was this

particular 2S albumin. Consequently, this transgenic soybean was never sold to the

general public, even though the modified soybean would have been harmless to the

majority of people who don’t experience Brazil-nut allergies.

The Starlink corn incident is another example of the government’s willingness to

protect people from any potential problem related to transgenic organisms. Starlink corn

was genetically modified with the Bt toxin Cry9C, which is produced by a different

subspecies of Bacillus thuringiensis that produces the more popularly used insecticide

Cry1Ac. The bacterial protein Cry1Ac and its closely related derivative had been used as

a topical insecticide for over 30 years for both traditional and organic crops, and in the

past decade the gene coding for it has been inserted in a growing number of crops such as

corn and potatoes. However, with such widespread use, it was feared that insects would

25

acquire a resistance to the toxin through a mutation of the toxin binding site in the insect

midgut. This was when Cry9C was introduced into corn, which binds to a different

binding spot than Cry1Ac [20], thus providing an additional line of defense in case of

insect resistance.

Initially the corn carrying the Cry9C protein— Starlink corn—was approved by

the USDA only for animal feed. Since Cry9c broke down more slowly than Cry1Ac, it

was feared that it would remain in the digestive tract of humans too long, therefore

increasing its risk of allergenicity. However, somehow this corn entered the general corn

grain supply of the United States. An activist group— the Genetic Engineering Food

Alert— did independent research and found trace amounts of the Cry9C protein in

several of the products it tested [21]. Although this protein had never been shown to

cause harm to humans, a large-scale response was launched that sampled over 110,000

different sources of grain [22]. A year later it was determined that consuming the corn

containing Cry9C had a low chance of affecting consumers, due partly to the fact that it

breaks down readily during processing [21]. However, it should be comforting to some

opponents of genetic engineering that there are agencies that are willing to take swift and

decisive action at the threat of harm.

Even if people are skeptical about the government’s efficacy about regulative

transgenic products, the fact still remains that traditional breeding methods introduce

allergens into food as well. If a farmer tries to selectively breed a new trait into any crop,

even if he is successful in adding that particular gene, undoubtedly a number of

unintended genes come along with it. Breeding two organisms together can never simply

transfer a single gene. Therefore, the chance that a new fruit or vegetable from a

26

nontransgenic crop will carry a protein that elicits an allergic reaction is much higher than

a genetically engineered product. With genetic engineering a single gene can be added.

And the product of that gene is probably well known before it is ever used to transform a

plant, so necessary and specific testing can be done to determine possible allergenicity.

Of course, the shelves of American grocery stores are lined with products that

elicit an allergic response in small segments of the population. A normal diet will

introduce many thousands of proteins, and only a few of these have been shown to

sensitize a person to an allergy [24]. If 2% of the people in America happen to have a

peanut allergy, does that mean peanut butter, mixed nuts, peanut butter cups, and the

remainder of products that contain peanuts should be pulled off the shelves? Of course

not. Transgenic foods are so stringently tested that there is virtually no chance that a

product will be introduced into the market that causes nearly the rate of allergies as do

peanuts.

Another common fear about genetically modified foods concerns the markers

used to test for transformation. When the public hears about the use of antibiotic

resistance genes, there is instantly a panic that somehow our last line of defense against

bacterial infections will be destroyed if these markers were ever to be picked up by

pathogenic bacteria. The mucosal surfaces of our bodies are homes to billions of bacteria,

most of which provide valuable services such as consuming nutrients so pathogenic

bacteria can’t grow. However, some of this normal microflora can become pathogenic if

for some reason its population suddenly increases. There are also pathogenic bacteria

mixed in this normal flora that will cause infections in instances such as a compromised

immune system. The fear is that if an antibiotic resistance gene is left in the final product

27

being consumed, that it could spread to pathogenic bacteria living along our digestive

tracts and lead to serious infections that are difficult to treat.

This isn’t a very realistic concern. First of all, antibiotic resistance markers can be

removed from transgenic plants, so the marker may never reach the human digestive

tract. Removal of markers will be discussed later in this paper. Also, commonly used

markers, such as for antibiotic resistance, will not pose a health risk if ingested by people.

DNA is genetic material of all organisms more complex than viruses, and any plant or

animal meat we eat will contain DNA. We can consume up to 1.0 grams of RNA and

DNA per day, which is a huge amount of genetic information [5]. However, if the marker

remains in the final product, the overall percentage of DNA from the novel genes is

infinitesimally small. Marker DNA will account for less than 1/250,000 of the total DNA

consumed [5]. Besides, the genes would have to survive the nucleases of the digestive

tract, and then bacterial cells in the digestive tract would have to be competent for DNA

uptake.

Even if the antibiotic resistance marker remained in the eaten product, if the

DNases fail to break it down, and if conditions happen to favor uptake of exogenous

DNA (these are a lot of ifs), markers that are most commonly used pose no threats to

human health. One of the most commonly used markers to test for transformation is npt

II, which confers resistance to the antibiotics kanamycin and neomycin [25]. This gene is

commonly present in many bacteria in the gut, so humans already have a high exposure

to the product of this gene. In fact, nptII was initially taken from microbes in the human

gut [25]. For this reason, kanamycin and neomycin are not prescribed as antibiotics.

Commonly prescribed antibiotics would never be used to test for transformation in plants

28

that were targeted for human consumption.

It’s clear that genetically modified organisms pose little threat to human health in

America, since there exists an elaborate infrastructure for testing any product to be

approved for human consumption. Transgenic products are even more stringently tested

than conventional foods, so they are not merely as safe to eat, but even safer in many

respects than unmodified foods. Also, the antibiotic resistance markers used to test for

transformation can be removed before the product reaches the grocery store, but even if

left in, poses an insignificant threat to human health. Beside, there are other markers

besides antibiotic resistance genes that can be used to test for transformation.

B) Improvements to Human Health: Genetic Engineering and Medicine

The potential benefits of genetic engineering are immense. Not only can it serve

to raise the quality of life in wealthy industrial countries, it can also provide nutrients and

medicines cheaply so that life may be possible in poorer countries that can’t afford the

rising costs of western medicine. The biosynthetic capabilities of plants and animals can

be harnessed to produce medicines and vaccines that are normally produced in

pharmaceutical laboratories. Using recombinant DNA technology to make medical

products is also cheaper in many respects, since larges amounts of proteins can be

synthesized at a relatively low cost, and storage expenses such as refrigeration can be

dramatically decreased. This is a particular advantage in poor countries in hot, humid

climates.

Plants can be used as factories to produce complex molecules in the correct

conformation for pharmaceutical activity. Since plants are eukaryotic cells, they contain

the endoplasmic reticulum and Golgi apparatus necessary for posttranslational

29

modifications. Also, some plants have chaperones that are homologous to those in

mammalian cells, which control the efficiency of protein assembly and the extent of

protein degradation [26]. Plants are even able to make molecules with several subunits

such as antibodies. Bacteria are somewhat limited in their ability to make complex

proteins for human use, since they lack the ability to perform many posttranslational

modifications necessary for the proteins to obtain their correct conformation.

Fig. 4

From Ma, Julian K.C et al. “Molecular farming for new drugs and vaccines: Current perspectives on the production of pharmaceuticals in transgenic plants.” European Molecular Biology Organization. 6:7. 593-599. (2005).

30

The use of genetic engineering to make products to promote human health and

prevent disease is more widespread than people recognize. There are over 2500 biotech

companies worldwide, with over 1300 in the US alone [6]. The result of this intensive

research and the billions of dollars invested is a myriad of products that people take every

day to prevent disease and serve to improve health. For instance, monoclonal antibodies

can be produced by genetically modified organisms that can aid in the prevention of

transplant rejection as well as serve as research tools. Recombinant clotting factors are

produced in mass quantities to prevent hemophilia. The list goes on and on.

In most cases the product of the transgenic organism, whether it be a plant, yeast,

or animal, produces a pharmaceutical product that is far superior to and causes far fewer

complications than the product obtained before modern genetic engineering. Bovine

insulin was once given to people with type II diabetes mellitus. However, the bovine

insulin caused allergic reactions in people due to the fact that bovine insulin differs from

human insulin by three amino acids [3]. Thanks to genetic engineering, scientists were

able to take pig insulin (which differs from human insulin by only one amino acid: it has

an alanine at position 30 instead of threonine [3]) and engineer it so that it is identical to

human insulin. Now potentially deadly allergic reactions are not a concern, and everyone

with type II diabetes can be treated with this recombinant product. Another example is

growth hormone, which used to be extracted from human corpses! It once took the

pituitary glands of 650 corpses to produce merely two to three grams of growth hormone

[3]. However, thanks to genetic engineering, microorganisms produce all the growth

hormone needed.

31

Perhaps the most intriguing possibility for genetic engineering is the use of a

cheap, stable, edible vaccine against some of the common diseases of our day. Plants are

suitable biosynthetic factories for the production of complex proteins in high amounts.

And plants are biosynthetic factories that can be eaten! As pointed out in ref. [27], many

“experiments demonstrated that plants can express, fold, assemble, and process foreign

antigens and can provide both a simple vaccine-manufacturing process as well as a

matrix for suitable oral immunization.”

Using plants as for the production of medicine is advantageous in affluent

countries like America, which could significantly decrease the costs of production. This

in turn can be passed along to the consumer as cheaper prices for drugs. However, the

countries that stand to benefit the most from having plants produce vaccines are the

poorer countries. Diseases that were eradicated decades ago in America, like polio,

continue to plague poor countries for a variety of reasons (including political and social

instabilities). One of the reasons is that only major pharmaceutical companies in affluent

countries possess the resources to mass-produce medications and vaccines. However, if

something as simple as a gene inserted into a common plant could produce the same

bioactive compounds created in American labs, then genetic engineering may potentially

be a relatively inexpensive way of vaccinating billions who otherwise wouldn’t have the

opportunity.

One particular disease that can successfully be vaccinated against with an edible

subunit vaccine is hepatitis B. As of 1996 the hepatitis B virus had infected 115 million

people worldwide, particularly in poor countries that can’t afford the current licenses for

32

the vaccines, or means of mass production and storage. In these countries hepatitis B is

endemic [27].

A means of rapidly and cheaply vaccinating large numbers of people is to get a

common edible plant, such as a potato, to express the surface antigen of the pathogen.

This way the immune system will recognize the foreign proteins of the subunit vaccine,

make antibodies and memory B lymphocytes in response, and leave the person with a

lasting immunity against a future infection of the actual pathogen. The most

advantageous aspect to having plants produce subunit vaccines is that no injections are

needed. The fruit or vegetable is consumed, and the antigens are processed like any other

protein eaten, ultimately getting absorbed through the mucosal layers of the small

intestine [27]. Amazingly these antigens survive the journey through the acidic

environment of the stomach and retain their necessary conformation. In 1996 121 million

Indian children were vaccinated against polio in just two days using oral vaccines [27].

Plants are not the only organisms that can produce complex, correctly folded

proteins for human use. Yeast cells also have amazing biosynthetic abilities. They also

possess the advantage of proliferating faster than plant cells, so large amounts of protein

can be produced relatively quickly. The disadvantage is that if the protein isn’t secreted

from the yeast cell, then it will have to be purified from the rest of the constituents of the

cell. The yeast Pichia pastoris can be used to make vaccines. P. pastoris is an excellent

expression system since it has an alcohol oxidase gene that is tightly controlled by its

promoter [28]. In the presence of methanol, the alcohol oxidase promoter is activated and

the gene downstream of it is transcribed.

33

Mutant strains of P. pastoris that can’t grow on minimal media can be used to test

for transformation. The initial transfer vector requires the transgene of interest, the gene

that codes for the biosynthetic enzyme lacking in the mutant strain (such as a histidinol

dehydrogenase gene if the strain is unable to synthesize histidine), and regions that share

sequence similarity with the region of insertion of the P. pastoris genome. A single

recombination event takes place where this cassette gets inserted, and the gene coding for

alcohol oxidase is removed. Only those P. pastoris cells that have been transformed will

grow on minimal media, and the product of the inserted gene, such as a subunit vaccine

for hepatitis B, will be produced with the addition of methanol.

Fig. 5: Production of Vaccine in Yeast

From Dominguez, Angel et al. “Non-conventional yeasts as hosts for heterologous protein production.” International Microbiology. 1:131-142. (1998).

Bacteria, yeasts, and plant cells can be used to make a variety of products that

have nutritional and therapeutic compounds for humans. Even animals can be used to

make proteins that are correctly folded and modified for human use. One example is the

product for the gene alpha-1-antitrypsin, which is lacking in people who have a heritable

form of emphysema. To make this protein a vector is constructed which contains the

alpha-1-antitrypsin gene next to the gene that controls milk production [29]. This vector

34

is then microinjected into fertilized sheep eggs, and then the eggs were placed back in the

mother sheep to develop like any other sheep [29]. In fact, the sheep will look and act

like any other sheep, only that it produces alpha-1-antitrypsin in its milk. These sheep are

absolutely normal, and are not harmed in any way by making this protein.

Allowing genetically modified organisms to produce pharmaceutical products is

an efficient and safe way to keep the ever increasing human population healthy. Many

therapeutic compounds are produced in this way already. In a world where non-

renewable resources such as fossil fuels are being consumed rapidly, it is nice to see a

technology that takes advantage of renewable sources to benefit the population. Once

again this production of pharmaceutical proteins is completely natural by nearly every

definition of the word, as we are merely using the biosynthetic capabilities of the

organisms around us to make a desired product.

35

IV) Genetic Engineering and the Environment

A) Environmental Benefits of Genetic Engineering

Genetic engineering can be used not only to enhance and sustain the health of

humans, but also enhance and sustain the health of our environment. By lowering the

amounts of herbicides and pesticides, by making crops less vulnerable to environmental

stresses, and by increasing the yields that a typical crop will produce, genetic engineering

has the potential to help every imaginable facet of agriculture. Since the advent of the

agrarian society, farmers have been seeking ways to protect their crops from pests,

droughts, floods—anything that could compromise their crops. Maintaining crops wasn’t

a luxury; it was necessary to survive. And society hasn’t changed all that much.

Agriculture is still the largest industry in most countries. With populations increasing at

an exponential rate, and with it the demand for food, it is necessary for agriculture to be

as efficient, productive, and environmentally friendly as possible.

Farmer have been fighting the battle against microorganisms and other pests since

the beginning of agriculture. The first recorded instance of pest control was 4000 years

ago, when Chinese farmers used ants to destroy leaf-eating insects [3]. Since then

pesticides have increased in their ability to kill insects, but also in their toxicities. A good

example is DDT, which targeted insects that were reducing annual crop yields. And it did

kill many species of insect pests. Unfortunately, it also killed many non-target insects,

even those that ate common pests of the crops, along with many species of birds like the

bald eagle which was almost driven to extinction by widespread use to DDT. Of course,

DDT is now illegal to use, but there are still toxic chemicals that are being used by the

ton that are seeping into water supplies, contaminating the soil, and having detrimental

36

health effects on any living creature in the areas where they are being used. Clearly the

indiscriminate dumping of harsh chemicals by methods such as crop-dusting is not the

most efficient or safe way to protect crops.

The environmental toll of using pesticides is only the beginning of the problem.

The human toll is also immense. “Malignancies linked to pesticides in case reports or

case-control studies include leukemia, neuroblastoma, Wilms' tumor, soft-tissue sarcoma,

Ewing's sarcoma, non-Hodgkin's lymphoma, and cancers of the brain, colorectal, and

testes” [30]. Organophosphates, which are a type of chemical insecticide currently in

use, were initially developed as chemical warfare agents that inhibit the enzyme

acetylcholinesterase and disrupt the function of cholinergic neurons [6]. This should

alarm some people, since our neuromuscular junctions are controlled by acetylcholine.

On the next page is a list of 46 chemicals typically used as pesticides which have

been shown to have carcinogenic effects by the International Agency for Research on

Cancer. Eight pesticides with sufficient evidence of carcinogenicity are still in use today,

with 15 more being strongly suspected as carcinogens [30]. It should be noted that other

countries may not adhere to American agricultural laws and may use compounds

outlawed here long ago. More alarmingly, the United States imports a large amount of

food from other countries, and we may be consuming large amounts of toxins that

continue to be used in other countries.

37

From Zahm, Shelia Hoar and Ward, Mary H. “Pesticides and Childhood Cancer.” Environmental Health Perspective 106(Suppl 3):893-908 (1998).

Fortunately, there are natural insecticides produced by bacteria that are extremely

effective in killing common pests in agriculture. “The delta-endotoxin proteins of B.

thuringiensis have been intensively studied, and no indications of mammalian toxicity

have been reported. Furthermore, approximately 176 different B. thuringiensis products

have been registered since 1961, and the regulatory agency [EPA] has not received any

reports of dietary toxicity attributable to their use” [31]. The gram-negative soil

bacterium Bacillus thuringiensis produces these insecticidal crystalline proteins (encoded

by Cry genes) during sporulation, and the spores can remain in a dormant state for long

38

periods of time when nutrients are scarce or environmental conditions are unfavorable for

survival. Presumably the Bacillus thuringiensis endospores will be consumed by the

insect, and the insect will subsequently die, leaving behind fresh tissue which the bacteria

can use as nutrient.

The Bt toxin is an ideal insecticide for agricultural use for a number of reasons.

First of all, there are a large number of subspecies of Bacillus thuringiensis, many of

which produce unique versions of the toxin which affect different types of insects.

Secondly, the toxin only stays active in the environment for a short time. Sunlight

degrades over 50% of the tryptophan residues of the parasporal crystal within 24 hours,

so the active toxin may exists in the environment for less than a day [6]. It may seem

strange to say that a substance’s fragility can actually be an attribute, but in this case it is.

The lack of persistence in the environment will drastically reduce the amount of toxin

affecting non-target organisms, while also decreasing the chance that a target insect will

acquire a resistance against the toxin. To go along with this point, it should be noted that

the Bt toxin is remarkably specific to its target insect, and has little or no effect on those

insects that aren’t pests to crops. This is a claim that can’t be made about synthetic

insecticides. At one time a hypothesis was made that various Bt toxins would wipe out

the monarch butterfly population, since the larvae consume milkweed which is found

predominantly around cornfields, and deposition of transgenic corn pollen can be found

on the surface of those milkweeds. However, this assertion was ultimately found to be

false, as purified Bt toxin was fed to butterflies and it had little detrimental effects on

them [32].

Butterflies aren’t the only organisms unaffected by the Bt toxins. Humans, cattle,

39

and wild animals living around sprayed crops would experience no direct harm by

widespread application. The toxin is ingested by insects and binds to specific receptors in

the gut only under alkaline conditions. The acidic environment and lack of specific

receptors in animals’ stomachs prevents the toxin from binding, and therefore it is

perfectly harmless.

This insecticide was so beneficial that approximately 90% of the microbiological

products designed to control insects are based on topically applied Bt toxin [33]. In fact,

since the toxins are naturally produced by bacteria, organic farmers have been using the

Bt toxin as an insecticide for over 35 years. As effective as these toxins were, however,

there was one major problem: topically applied toxin could not affect those species of

insect that burrow into the tissue of the plant. Insects like the Southwestern and European

cornborers were impervious to the Bt toxins, no matter how much was applied.

The solution to this problem is genetic engineering. By using the A. tumefaciens

Ti vector system, the gene or multiple genes coding for Bt toxins can be placed under the

control of a strong promoter (such as the 35S promoter from the cauliflower mosaic

virus), terminator and polyadenylation sequences (typically from the nopaline synthase

gene), and inserted directly into the genome of the crop to be transformed. This way the

Bt toxin will be produced in the tissue of the plant as well as on the surface, so all ranges

of insects susceptible to the toxin will be killed. Fusion proteins can also be made by

putting two or more segments of different Cry genes together under the same promoter to

make a novel protein with an increased range of effectiveness.

The amazing flexibility to manipulate genes through genetic engineering provides

ways to increase the amount and type of toxin produced, as well as a means to prevent

40

insects from acquiring resistance to the toxin. Large amounts of the toxins are used, both

by transgenic crops and by topical application, and this widespread use puts tremendous

selective pressure on the susceptible insects to evolve resistance. Nature certainly isn’t

static. Any act to suppress a certain species will lower the population initially, but those

mutant members that, by chance, are not susceptible to the treatment will survive, and it

is only a matter of time before they build up the population with strictly resistant

organisms. In the case of the Bt toxins, tolerance has been acquired by insects through

alterations in the insect’s midgut receptors that prevents the toxin from binding and

causing its physiological effect [34]. It only took 1-2 years of widespread use of the Bt

toxin for mosquitoes to evolve a resistance in some tropical countries [35].

It is this resistance argument that is most commonly used to argue against genetic

engineering. However, it often seems like people making this argument assume that

scientists are powerless in the face of nature, that these problems that science runs into

can’t be addressed by science. Evolution is not idly sitting by and letting scientist do what

they want with the biological world, so it is unfair to expect scientist to sit by passively

and not act to fix any problems that modern technology may come across. There will

always be a push and pull, a move and a countermove when dealing with nature. An

action is taken, and life molds itself around that action. There will never be a technology

that perfectly and permanently fixes a problem. However, science can always address

problems that may arise. If the insect’s midgut receptors change, then scientists can

always alter the toxin to bind once again.

Scientists have created a novel protein that causes the same pharmacological

effects as the regular Bt toxin, but also has an additional, non-native polypeptide at its

41

carboxy-terminus that binds with a greater affinity to toxin receptor in the insect’s midgut

[33]. It is necessary for the toxin to bind to its specific receptor to have its toxological

effect, which is creating pores in the insect’s gut. It has been discovered that N-acetyl

galactosamine residues on this receptor are an important component for binding of the

toxin to the receptor [33]. Ricin-B is a galactose and N-acetyl galactosamine specific

lectin that binds to these specific residues with a high efficiency, so adding the gene

coding for Ricin-B to the Cry1Ac gene will create a protein with a very high affinity for

the receptor. This new protein not only killed a greater percentage of insects that are

normally susceptible to the Bt toxins (due to the increased binding affinity), but also

affected a wider range of insects than those that are normally susceptible to the toxin

[33]. Now it would most likely take a mutation of two different genes simultaneously to

fully prevent toxin binding, which is unlikely, and therefore resistance is prevented.

The important aspect of this experiment is not that Ricin-B binds with a high

affinity to the insect receptor, since there are many galactose-specific lectins that can be

used besides Ricin-B (and probably more appealing than Ricin-B). Rather, this

experiment shows that measures can be taken to combat possible resistances to transgenic

crops. If by chance the toxin’s receptor undergoes a mutation which allows the insect to

tolerate the Bt toxin, then scientists can study the new structure, and make adjustments by

merely changing the binding characteristics of the protein produced. It is possible that in

the near future other insect receptors can be targeted, perhaps with a toxin other than Bt.

The possibilities are endless.

Besides, there are already strategies to combat insect resistance, such as refugia,

which requires that a certain proportion of the total acreage of land be set aside for non-

42

transgenic crops. This way the non-susceptible insects will be able to mate with those

susceptible to the toxin, and the genes that make insects vulnerable to the toxin will

remain in the gene pool.

The increasingly widespread use of transgenic Bt crops against several prominent

pests has had positive effects for the environment. A four-year agriculture research study

sponsored by the USDA measured pesticide runoff into the Mississippi river, which

flows through a 7000 square mile cotton producing area. “The fewer pyrethroid

applications needed on Bt cotton sites reduced the amount of pesticides released into the

environment. And while runoff from non-Bt cotton sites contained very slight amounts of

pyrethroid insecticides, runoff from Bt cotton sites had almost none at all” [36].

As the population increases genetic engineering will become a necessary facet of

agriculture. More crops will be needed, and this means more pesticides and herbicides

will be required to protect the crops, which means that an increasing amounts of

chemicals toxic to humans and animals will enter the environment. A smarter way of

producing food for the world is needed, and genetic engineering can do this in a number

of ways. Genetic engineering is already a part of all of our lives. As of 1999, 40% of

corn, 45% of soybeans, and 50% of cotton produced in the United States came from

genetically modified crops [35]. Now genetically modified plants cover 131 million acres

of American soil [35]. Clearly these transgenic products are safe, and the advantages to

agricultural production and sustainability that genetic engineering provides will only

increase in the future.

43

B) Addrssing Fears about Biotechnology and the Environment Fears about possible allergic reactions to new proteins added into food have

already been addressed in previous chapters. Also, the concern about antibiotic resistance

markers affecting soil bacteria has been touched on. However, since the potential creation

of “superbugs” is a common fear that persists in many people’s minds, it is necessary to

explain why the creation of antibiotic resistant soil bacteria does not warrant too much

concern.

The United States Food and Drug Administration stated in 1995 that horizontal

gene transfer does not occur between plants and bacteria [37]. However, this might be

something of an overstatement. Nucleotide sequences in databases have shown that some

genes in soil bacteria come from plants [38]. Therefore, there must be some mechanism

for horizontal gene transfer between plants and bacteria, even if it occurs infrequently

under very specific conditions.

While there are well known cases of gene transfer from a bacterium such as A.

tumefaciens to a plant and from viruses to plants, it appears that the transfer of a gene

between a eukaryotic plant and a prokaryotic bacterium faces many barriers if it can

happen at all. One likely way that this would occur is through transformation: the loss of

naked DNA from the plant and the uptake of that DNA by a bacterium. Several

conditions must be met in order for this to occur. “These conditions include release of

DNA very close to those few bacteria that have the relevant molecular mechanisms and

which actually develop the physiological state of competence. The naked DNA must also

escape any rapid chemical and enzymatic degradation and avoid being irreversibly

adsorbed onto soil components” [39]. The DNA sequence in question also must be

44

integrated into the bacterial genome. For this to happen, there needs to be sequence

similarity between the plant gene and a portion of the soil bacterium genome. There also

needs to be a promoter that comes along with the plant gene that works effectively in a

prokaryotic cell.

Many experiments have tried unsuccessfully to simulate gene transfer between a

plant and a bacterium. Neilson et al. tried to accomplish gene transfer in non-sterile soil,

claiming that if it happens at all, it occurs at a frequency of once in every 1010 to 1011

bacterial cells, which is below the level of detection [40]. Clearly this occurrence under

field conditions is a rare event. One obvious reason for this is sheer probability. The

native plant genome is vast compared to the antibiotic resistance gene and any other

transgenes that were added, so transfer of these select genes will be infrequent. It was

hypothesized that as the complexity of the donor DNA increases, the frequency of

transformation for a given gene decreases [39].

The bottom line is that the number of physiological events that need to occur in

order to allow transformation of a soil bacterium by plant DNA makes this phenomenon

rare. If one is to take action to prevent the creation of superbugs that are resistant to all

commonly prescribed forms of antibiotic, then genetic engineering is a poor place to

focus one’s energies. Rather, the haphazard use of antibiotics in the beef and milk

industries, as well as physicians’ propensity to prescribe antibiotics far too often are more

logical places to start. Furthermore, bacteria are naturally resistant to many of the

antibiotics that are used by humans [1], which has absolutely nothing to do with modern

genetic engineering at all.

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There are several options if one desires to remove the antibiotic resistance marker

from an edible plant. One way is to co-transform the desired plant with two separate

plasmids: one carrying the marker gene, and the other one carrying the gene of interest

into the plant. These genes will integrate into different portions of the host’s genome, and

then traditional selective breeding can produce offspring which contain the gene of

interest, but not the marker gene.

Another way to remove the marker is through site-specific recombination through

the Cre/loxP system. The marker gene is flanked by specific DNA sequences called the

loxP recombination sites, and Cre is the enzyme that cuts out anything between these two

sites after it has been inserted into chromosomal DNA. The gene coding for the Cre

enzyme is located on its own plasmid and is controlled by a specifically induced

promoter. In one experiment, the XVE system was used, which is induced by the addition

of estradiol [45]. When estradiol is added, the antibiotic resistance marker was removed,

activating a GFP gene that will be the evidence that the marker has been removed [45].

This way the antibiotic resistance marker can be removed, and the desired gene remains

to have the designed effect.

Analogous to the fear of antibiotic resistance markers spreading to soil bacteria is

the fear that herbicide resistance will spread to weeds, creating a weed that grows out of

control and destroys crops. Pollen from transgenic crop can travel over long distances,

even be carried across international boarders and germinate in countries that never

intended to harvest transgenic crops. A perfect example are the mountains of Oaxaca,

Mexico, where the world’s greatest genetic variety of maize is maintained in open-

pollinated fields [41]. These fields are a great source of historic and cultural pride for the

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Mexican people, and it was never intended for transgenes to make their way into these

fields. In fact, Mexico outlawed the planting of genetically engineered maize within its

borders in 1998 [42].

However, a 2001 article in Nature claims that transgenes were found in some

species of corn in Oaxaca [42]. A 35S promoter sequence (from the cauliflower mosaic

virus) was discovered in kernel samples and in grain sold in local stores [42]. The 35S

promoter is a strong and constitutive promoter that is commonly used to drive the

expression of an inserted transgene, so it is a logical target to test for transgenic

contamination in species. Also, a nopaline synthase terminator sequence and a Bt

Cry1AB endotoxin sequence from Bacillus thuringiensis was detected in some samples

of maize [42]. Of course, these genes could have never appeared in wild species of maize

unless the Oaxaca fields were specifically contaminated with genes from transgenic

plants. It is possible that Mexican farmers may have unknowingly planted transgenic

kernels within their borders.

However, a more recent publication in Nature claims that the Oaxaca maize was

uncontaminated. The seeds of 870 plants from 125 different fields in Oaxaca were

screened for the CaMV 35S promoter and the nopaline synthase terminator. Neither one

of these genes were found in the maize [41]. Certainly, more research needs to be

conducted on the maize in these remote Mexican fields.

What is clear is that pollen can potentially carry transgenes into lands where they

are not welcome, just as pollen can carry any gene. In fact, it’s difficult to conceive how

the spread of transgenes can be stopped, even to remote mountainous lands like Oaxaca,

Mexico. With trade occurring freely between America —which is the world’s leader in

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producing genetically modified organism— and the rest of the world, it is impossible to

prevent these transgenes from crossing borders. The United States exports several million

tons of maize to Mexico alone every year (6.5 million tons in 2001 alone), some

transgenic and some conventional [43]. Mexican farmers could easily plant these in their

own fields, unknowing whether they are planting transgenic or conventional kernels—

perhaps not even knowing that such a thing as a transgenic plant exists.

It is unfortunate that the genes for the Bt toxin contaminated Mexican maize.

However, the spread of transgenes can have more dramatic consequences than merely

offending cultural pride. A much more harmful prospect is that a gene for herbicide

tolerance will end up in the wild population, perhaps in a weed, creating a superweed that

will be difficult to control. This is not as far fetched as it seems. “Of the 60 crop plants

under widespread cultivation, at least 49 have wild and often weedy relatives to whom

they have the potential to cross” [35]. The same mechanisms that allows plants to spread

desirable traits considerable distances is the same mechanism that will allow transgenes

to spread rapidly if it creates some sort of selective advantage. This is how plants

evolved. The risk of crop gene flow to weedy relatives has always existed, and such

‘gene flow’ occurs where possible. The risk of gene transfer to weeds is similar with both

conventional and GM crops and is not contingent on how we introduce these genes into

plant” [4]. And pollen can spread genes over long distances. Separating different maize

varieties by two-hundred meters—the length of two football fields—will result in

contamination due to cross pollination of pure plants of about 0.1% [44].

With the number of different crops in widespread cultivation, and the sheer

volume of international trade, the unintended spread of transgenes is inevitable. However,

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once again scientists aren’t helpless against this undesired consequence of genetic

engineering. Due to our ever-increasing knowledge of the molecular world, there are

ways to track and even prevent the spread of transgenes. Scientists aren’t powerless to

prevent unwanted gene flow, however. One intriguing way to track the spread of

transgenes is to use a marker, such as green fluorescent protein. This is used extensively

in research laboratories, and can easily be used to mark only those plants that possess the

transgene in them. This way the farmers of the mountains of Oaxaca, Mexico could tell

which maize crops were of native origin and which have been contaminated with the

transgene.

Fig 7: Detection of transgenic seeds using red fluorescent protein as a visible marker. Maize and tobacco seeds (A and B) observed under green light (C and D).

From: Ma, Julian K.C et al. (2005) “Molecular farming for new drugs and vaccines: Current perspectives on the production of pharmaceuticals in transgenic plants.” European Molecular Biology Organization. 6:7. 593-599.

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If the prospect of eating a glowing tomato is a little too strange to bear, there are

precautions that can be taken to severely limit the chance that a transgene will spread to a

related plant. One interesting solution is to insert the transgene into a plastid, such as the

chloroplasts in green photosynthetic plants. Chloroplasts contain their own distinct DNA

that is separate from the chromosomal DNA, since chloroplasts, like the mitochondria in

virtually all eukaryotes, were once free living organisms that evolved to have a symbiotic

relationship in eukaryotic cells. The advantage of inserting the gene of interest into the

chloroplast genome is that its DNA is inherited in a non-Mendelian fashion. In most

angiosperms, the plastids are maintained solely in the egg cell during reproduction, so it

is transmitted only by the female [46]. This means that the pollen will not contain any

genetic information concerning the plastids, and therefore any transgenes contained in the

plastid cannot be spread to distant, related plants.

Another major benefit of using plastids as a carrier of transgenes is that they are

present in such high numbers in a plant. “A chloroplast gene will usually be present

thousands of times per cell with the result that chloroplast genes produce the most

abundant proteins on the planet” [47]. With many chloroplasts per cell, and many copies

of chloroplast DNA per chloroplast, any gene inserted into the chloroplast genome will

be expressed at high levels, thus producing large amounts of protein.

However, a potential dilemma exists. If plastids were once free-living organisms

that learned to rely on another cell to live, and therefore lost most of their genes to the

larger nuclear genome, then it was obviously possible for genes to escape the plastid into

the nuclear DNA. This seems to be the case, but at a very low level. Plastid inheritance

was studied over a 3 year period of time between wild populations growing next to

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Figure 8: List of potential solutions to the gene flow of transgene

Technique Advantages Disadvantages Maternal inheritance Prevents gene flow through out- Techniques to export crossing and volunteer seeds. Field proteins are not yet tests indicate low incidence of available. sympantry and mixed stands extinct in three years. High levels of trans- gene expression and no evidence of gene silencing or position effects. Male Sterility Prevents outcrossing. Shelf-life of Crops need to be propagated Flowers may also be extended. By cross-pollination from Several tapetup specific non-GM crop or by promoters available. artificial seed. Potential for volunteer seed dispersal Seed Sterility Controls both outcrossing and If transgene is silenced, volunteer seed dispersal introgression will occur. All linked genes should segregate together Cleistogamy Pollination occurs before flower In practice, introgression opens, theoretically preventing occurs despite self- outcrossing pollination

Apomixis Seed is of vegetative origin and Only known in a few crops. not from sexual cross. Controls Genes not yet available. both outcrossing and volunteer seed dispersal. Hybrid traits can be fixed Incompatible genomes Prevents recombination after May not be applicable pollination to crops that exhibit homologous recombination. Crops will not produce seeds unless propagated with compatible plants Temporal and tissue- Gene either activates only when May not be applicable to specific control via product is necessary or excised traits required throughout inducible promoters before flowering the plant’s life. If chemical treatment fails to penetrate plant tissue, residual level of transgene may be present in pollen or seeds that could be outcrossed.

Modified from Daniell, Henry. “Molecular strategies for gene containment in transgenic crops.” Nature Biotechnology. 20. 581-586. (2002).

51

oilseed rape, and it was discovered that gene flow was extremely rare between the

transgenic and the weed species, even though some of the weeds were relatives of the

oilseed rape [48]. This means that a transgene placed in the chloroplast of a plant will be

safely retained in the chloroplast, preventing unwanted spread of the transgene.

Another potential solution to prevent the unwanted spread of genes is to make the

gene only active under specific conditions, such as the application of a particular

chemical. This way the transgene will be transcribed only when the farmer wants it to,

and silent in those weeds and crops that the transgene wasn’t intended for. One such

system used ethanol vapor to successfully induce genes in tobacco, potatoes, and oilseed

rape [49]. This system uses the alc expression system, which acts as a switch, turning on

gene expression for any gene fused to the modified alc promoter [49]. This system allows

the transcription of a gene under very low concentrations of ethanol vapor (as low as 0.4

µM of ethanol vapor). Again, this experiment is important in that it shows that transgenes

can be selectively transcribed, so even if a Bt toxin gene finds its way into a distant corn

field due to travel of pollen, that the gene won’t be expressed in those fields unless the

particular inducer is supplied. In the future other inducible systems can be devised that

will be activated by a cheap compound that can be sprinkled onto crops, or added to

water and taken up by the roots.

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VI) Bioterrorism So far this paper has dealt with unintended consequences that biotechnology may

encounter due to natural mechanisms. However, there remains one major fear that many

people are concerned about: the misuse of biotechnology to intentionally harm other

people. Although it is not the fault of genetic engineering if someone uses it to harm

others, just as it is not the gun’s fault if a person shoots another person, it is a topic that

has received a lot of attention in this post 9/11 era. It is possible to alter a microorganism

to create a biological weapon, although it is not as simple as many people may believe,

since it takes a certain level of expertise and resources that the average person simply

doesn’t possess. However, the mere possibility of bioterrorism warrants at least a mention

in an honest discussion of genetic engineering.

The use of biological agents as weapons is not a novel notion. “Mongol invaders

catapulted plague victims into besieged cities, probably causing the first plague epidemic

in Europe, and British settlers distributed smallpox-infected blankets to Native

Americans” [50]. This isn’t an instance of engineered organisms through the use of

recombinant DNA, but it shows how pathogenic microorganisms can cause far-reaching

devastation (and it is yet another argument against the theory that natural is always good).

Unfortunately, the desire to harm others with the use of pathogenic microorganisms

persists in recent times. During the cold war the Soviet Union reportedly introduced alien

genes into a strain of Bacillus anthracis to increase its pathogenicity [35]. In addition, the

USSR’s Biopreparat agency reported stockpiles of a wide range of potential deadly

microorganisms, including hundreds of tons of anthrax and 20 tons of smallpox [35].

More recently, the Japanese Cult Aum Shinrikyo—the group that attempted a nerve gas

53

attack on a Tokyo subway—attempted to obtain Ebola virus during an outbreak in Zaire

in the 1990’s [39].

The constant advances in our understanding of the molecular world have given

scientists powers that were once unthought-of just decades before. For instance, in 2002

the poliovirus was synthesized in a lab without using genetic material derived from a

natural virus [51]. This was the first time that a virus fully capable of infection was

created de novo in a laboratory from scratch. Since the genome of the poliovirus is

available on-line, specific oligonucleotide sequences can be ligated end to end in a

laboratory. The virus was then injected intracerebrally into mice and had the same effect

as the wild poliovirus, with the only difference that the synthetic poliovirus required

larger doses to cause flaccid paralysis or death [51]. Of course, most of the world has

been vaccinated against poliovirus, so it isn’t a likely weapon for bioterrorists. However,

the fact that a viral genome can be assembled in a laboratory is enough to cause concern

that a more virulent microorganism can soon be constructed.

The virus that has received most of the public’s fear lately is smallpox.

Fortunately, there are a couple of reasons why smallpox would be more difficult to

construct in a lab from scratch. First of all, its genome is much larger, consisting of about

200,000 base pairs, as opposed to the 75,000 base pairs of the poliovirus [50]. Secondly,

the smallpox genome is not published for the general scientific community to see.

Perhaps most importantly, the World Health Organization prohibits DNA recombination

studies between variola (virus that causes smallpox) and other members of the

orthopoxvirus family [52].

54

Smallpox stirs such widespread fear since very few people in the population have

actually been immunized against it. For this reason, there are supposedly only two

stockpiles of the virus that remain in the world—one in the United States and one in the

former Soviet Union [35]—each under tight security so a potential theft is unlikely.

However, another route to reintroduce smallpox it to introduce the virulence factors of

the variola virus into a more common organism. These virulence genes can be introduced

into vaccinia, which is related to variola (the virus that causes smallpox) but harmless to

humans. This is exactly what has been done at the University of Pennsylvania.

The complete variola genome may not be published to the general scientific

community, but the genes, locations, and sequences that code for the virus’ virulence

factors has been published for anyone to see [52]. Both the vaccinia and the variola virus

cause the cells they infect to make proteins that inhibit pivotal components of the immune

complement system. However, the difference between the two is that variola causes

infected cells to make smallpox inhibitor of complement enzyme (SPICE), and vaccinia

causes infected cells to make a homologous protein called the vaccinia virus complement

control protein [52]. Both viral gene products inhibit the immune complement system,

but SPICE from variola is 100 times more efficient than the VCP of vaccinia, so variola

is deadly to most humans, and vaccinia can only cause problems in immune-

compromised individuals [52].

There is only an 11 amino acid difference between the VCP protein that is so

harmless that it can be used as a live vaccine, and the deadly SPICE protein that causes a

30-40% fatality rate for those infected with smallpox [52]. Even more frightening, this

research team from the University of Pennsylvania used site-directed mutagenesis on

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only 13 VCP nucleotide base pairs and created the gene that encodes the deadly SPICE

protein [52].

The ramification of this type of work are frightening. A relatively innate, easy to

acquire vaccine such as vaccinia can be turned into a potential biological agent. This is

perhaps the most frightening potential that genetic engineering has unlocked: that a

harmless microorganism can be engineered to be highly pathogenic and resistant to

modern medical care.

Figure 9: Comparison between SPICE from variola and VCP from vaccinia. In A, arrows point to the amino acids that are different between the two. B is a schematic view of SPICE mapped onto VCP.

From Rosengard, A., Liu, Y., Nie, Z., Jimenez, R. ‘‘Variola virus immune evasion design: Expression of a highly efficient inhibitor of human complement.” PNAS. 99:13, 8808-8813. 2002.

Most of the problems that may arise due to genetic engineering have relatively

simple scientific solutions. The ease of fixing some of these potential issues, such as the

unintended flow of genes, should be encouraging to those who are skeptical of

widespread use of this burgeoning technology. The ingenuity of modern scientists should

not be underestimated, and it can be trusted that if unforeseen problems do arise, that they

can be fixed in one way or another. Other problems, such as the spread of markers to soil

bacteria, are not serious and should not halt progress of genetic engineering. The only

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problems that are outside the control of scientific intervention are the ill intentions of

those people who want to do harm to others. This is not so much a scientific issue as it is

a social and political issue. Perhaps the most assuring point to be made about this is that

the potential to unleash smallpox on a population has existed for some time, and yet mass

bioterrorism has yet to be actualized. It is probably more rational to go on with scientific

advancement and not underestimate the humanity of others, even our enemies.

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VI) Conclusion Genetic engineering is the extension of methods and technologies that humans

have been building on for thousands of years in order to survive. This world would be

incompatible for civilized life if prehistoric humans hadn’t initiated the process of

guiding the world around them. The major crops we consume for nourishment everyday

were once small, wild weeds that were incapable of sustaining more than a small group of

people for an extended period of time. These weeds were plucked out of the forests,

selectively bred to grow larger and faster in non-native environments, and gradually were

directed into the plentiful crops we have today. The same process was repeated to

domesticate wild animals. This was all accomplished through genetic engineering: the

intentional manipulation of organisms on a genetic level to serve a particular function.

The only difference between the genetic engineering of 5,000 years ago and today is that

our techniques are more refined. However, the desire is the same. The need is the same.

Societies can only be built around a stable environment, but the environment will only

possess some semblance of stability if humans attempt to exert a measure of control over

it. Quite simply there are many forces in this world that don’t particularly care if humans

survive or not. Genetic engineering can better protect and enhance our food sources,

provide plentiful and easy to administer vaccines, and provide pharmaceutical products

that are cheap to produce and store so that even impoverished countries can afford quality

medical care. Forfeiting our most powerful technology to enhance and protect human life

and provide a sustainable environment is sheer insanity.

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